Directed neuronal migration is an essential feature of the developing nervous system, requiring the precise navigation of cells through a complex and dynamic molecular environment. Errors in this process can give rise to severe anatomical and cognitive disorders in human development. Although some of the genes affected in these defects are known, the molecular mechanisms that guide migration remain poorly understood, in part due to the complexity of vertebrate preparations. This issue can be addressed in the enteric nervous system (ENS) of the moth Manduca, in which an identified set of migratory neurons (the EP cells) and their pathways (visceral muscle bands) remain uniquely accessible throughout development. Despite obvious morphological differences, neuronal development in this system employs similar signaling processes as found in mammalian systems, permitting a mechanistic analysis of migration in living embryos. Specifically, fasciclin II (MFas II) has been identified as an essential guidance cue for the EP cells. Like its vertebrate counterpart NCAM, MFas Il is a member of the immunoglobulin related adhesion receptor family. MFas II is required for normal migration, and its two isoforms (transmembrane and GPI-linked) exhibit distinct patterns of expression in the migratory neurons and their pathways. Intracellular injections of isoform-specific RNA and plasmids will be used to induce ectopic MFas II expression in non-pathway muscles, while antisense oligonucleotides and RNAi probes will be injected to block endogenous MFas II expression to test whether this receptor is both necessary and sufficient to support migration. The migratory behavior of the neurons will also be tested in primary culture on MFas Il-transfected cells. Parallel experiments will be used to test how each isoform affects neuronal motility. One of the isoforms has been shown to associate with a tyrosine kinase, whose identity and function will be determined by co-immunoprecipitation, microsequencing, and manipulations in culture. Column-linked GST fusion proteins and 2-hybrid approaches will be used in function-independent screens of MFas Il-interacting proteins. These experiments should lend insight into the fundamental mechanisms by which this important class of guidance cues regulate neuronal migration in vivo.